JP2010500302A - Stable and bioavailable composition of lycopene isomers for skin and hair - Google Patents

Abstract

The present invention relates to a method for producing a stable composition rich in cis-lycopene (Z-form isomer) by heating tomato, a part of tomato, a derivative thereof, or a tomato extract in a solvent in a solvent for a long time. .

Description

  The present invention relates to a primary composition comprising at least one lycopene-containing material enriched in the Z-isomer of a lycopene compound with enhanced stability and bioavailability, and a method for its production. The invention also relates to an oral composition comprising the primary composition in a food, food supplement, cosmetic formulation or pharmaceutical formulation.

  The absorption of carotenoids is released from the microstructure matrix of food, dissolved in mixed micelles, intestinal uptake, uptake into chylomicrons, tissue dispersion, uptake in the liver, and gradually converted to LDL It is a complex process involving re-secretion into.

  Lycopene absorption from food sources is widely described in the literature. Lycopene bioavailability from foods such as tomatoes and tomato juice is very low. At present, tomato paste is the best known food source for bioavailable lycopene. Tomatoes contain more than about 90% of all E-type lycopene.

  Although tomato extracts containing large amounts of lycopene are commercially available in the form of oleoresin, from these sources the bioavailability of lycopene in humans is considerably limited. In concentrated tomato extracts, lycopene is present mainly in the crystalline state, which was said to be one of the main factors that reduce its bioavailability.

  At present, the most commercially available lycopene sources, whether they are derivatives (eg sauces) or extracts, exhibit an isomer profile that closely resembles the starting tomato, or a small amount of the Z isomer Only an increase is shown. For example, many treatments as thermal processing are known to promote isomerization, and Shi et al., Journal of Food Process Engineering 2003, 25, 485-498 describes Z-type isomerism by heating tomato sauce. It has been shown that the body can be increased. However, certain lycopene isomers are not stable and tend to reverse isomerize. According to the literature, the 5-Z form is the most stable of the major lycopene isomers, followed by the all-E, 9-Z and 13-Z forms. Thus, the stability of an isomerized lycopene-based product depends on its lycopene isomer profile and can be adjusted by processing techniques that affect this profile.

  Thermal isomerization of lycopene is known to improve its bioavailability from food matrices. However, the bioavailability of individual lycopene isomers has not been studied so far. With respect to stability, it can be considered that the bioavailability of a lycopene-based product depends on its lycopene isomer profile and can therefore be adjusted by technical means.

  There are already patents proposing technical means and compositions for improved bioavailability of lycopene. For example, WO 2005/075575 provides a primary composition enriched in the Z isomer that is effective in increasing the bioavailability of lycopene.

  EP 1 103 579 discloses a method for extracting lycopene by refluxing ethanol, which is carried out for a short time (30 seconds) so that no significant isomerization takes place. In practice, natural all-Z lycopene is desirable as the final product.

  WO 96/13178 discloses a process for preparing a stable lycopene concentrate. However, the effect of isomerization on stability is not recognized and, on the contrary, the disclosed method attempts to avoid isomerization by using temperatures below 50 ° C. for several minutes, for example 10 minutes.

  EP 1 201 762 refers to lycopene-containing products in which lycopene is in its natural all-trans form. Any heat treatment (dissolution and concentration) takes place in the shortest possible time (less than 1 hour) so that no significant isomerization occurs.

  KR 2005 006592 discloses the preparation of lycopene-zinc complexes with increased antioxidant activity. Heat is applied in the absence of solvent to break the cell walls of the plant material. Heating for a long time is not performed and isomerization does not occur.

  WO 03/0779816 discloses a method for preparing a tomato extract with a high lycopene content, wherein the extraction is carried out at room temperature.

  US 5,837,311 and WO 97/48287 disclose a process for preparing an oleoresin having a high lycopene content and satisfactory stability. The latter feature is obtained by phospholipids and glycerides present in the oleoresin itself. There is no mention of lycopene isomeric composition and its effect on stability. Hot extraction is performed to maximize the yield of lycopene, but not exceeding 1.2 hours. Only to improve the pulp-liquid separation, the tomatoes are heat pretreated in the absence of solvent.

  WO 2005/075575 mentions the problem of increasing the cis isomer content in tomato oleoresin: isomerization is carried out in particular by a short heat treatment. Furthermore, the resulting product contains a large amount of unstable 13-cis isomer.

  EP 0 937 412 discloses a method for preparing a finely divided powdered carotenoid formulation comprising heating for a very short time (5 seconds) in the presence of a solvent.

  US 5,858,700 relates to a method for isolating and purifying lycopene crystals, characterized by hydrolyzing impurities such as glycerides and phosphonates. The hydrolysis is carried out at a high temperature, but for a time shorter than 2 hours, and in some cases the extraction step using refluxing solvent is performed first, but is always performed for a short time to prevent lycopene degradation.

  US 6,235,315 discloses a stable powdery lycopene composition, characterized in that lycopene has a certain degree of crystallinity and has the lowest possible degree of isomerization.

  WO 03/090554 discloses a concentrated tomato derivative having a high lycopene content and having a predetermined viscosity and sugar content. This method is physical and does not use a solvent. In order to facilitate the separation of the pulp, the whole tomato is heated for 1 to 6 minutes.

  Mayer-Miebach et al., "Thermal processing of carrots: lycopene stability and isomerisation with regard to antioxidant potential", Food Research International, Elsevier Applied Science, Barking, GB, vol. 38, no. 8-9, October 2005, pages 1103- 1108 investigates isomerization as a function of temperature of lycopene in lyophilized carrots. The authors conclude that only the all-trans isomer is stable to prolonged heating and the cis isomer decreases.

  Indeed, the prior art provides a method for isomerizing lycopene, comprising obtaining a product with a highly bioavailable and stable isomer composition by heating in an organic solvent for a long time. Not done.

  The stability of individual Z-type lycopene isomers varies between isomers, in particular 13-Z lycopene is much less stable than 5-Z or 9-Z or all-E isomers. I understood. As a result, the primary composition of the present invention should have as little content of the 13-Z isomer as possible in order to exhibit optimal stability. Some Z-form isomers of lycopene (eg, 5-Z and 9-Z) have also been shown to improve the bioavailability of compositions containing lycopene. Accordingly, the primary composition can be used as a major component to provide improved bioavailability and biological effects as 5-Z isomers, or 9-Z isomers and 5-Z isomers. Should include combinations with the body.

  That is, the first object of the present invention is enriched in a specific mixture of Z-form isomers of lycopene, wherein the weight percentage of the isomer selected from the group consisting of 5-Z, 9-Z and combinations thereof is 13- The object is to provide a primary composition comprising at least one lycopene-containing material which is larger than the Z-isomer.

  In one embodiment, the present invention provides an oral composition comprising the primary composition in a food, food supplement, cosmetic formulation or pharmaceutical formulation.

  In one embodiment, the present invention provides a primary composition as an additive in a food for oral administration such as a nutritional composition, food supplement, pet food product, cosmetic formulation or pharmaceutical formulation.

  In one embodiment, the present invention provides a method for producing the primary composition or a food supplement, cosmetic formulation or pharmaceutical formulation comprising the same.

  In another embodiment, the present invention is as described above for the preparation of an oral pharmaceutical or cosmetic composition intended for improving skin health, in particular for protecting against skin photoprotection or skin tissue aging. Use of a primary composition.

  In another aspect, the present invention provides the use of the primary composition for the preparation of an oral pharmaceutical composition or cosmetic composition for preventing or treating cardiovascular disease or cancer.

  An advantage of the present invention is to provide a composition of the Z-isomer of lycopene that exhibits high stability, bioavailability and biological effectiveness.

  Additional features and advantages are described herein and will be apparent from the following detailed description and drawings.

Tomato paste (all E-type lycopene), tomato oleoresin rich in 5-Z lycopene (5-Z oleoresin), tomato oleoresin rich in 13-Z lycopene (13-Z oleoresin) or 9 and 13-Z Graph showing the area under the curve (AUC) of plasma lycopene / triglycerides of TRL after consumption of a standard diet containing a total of 25 mg of lycopene from tomato oleoresin rich in type lycopene (9- & 13-Z oleoresin) It is.

  The present invention relates generally to compositions that provide health benefits. More particularly, the present invention relates to beneficial nutritional compositions that can be used to improve skin and hair, and methods related thereto.

  The present invention makes available to consumers improved compositions obtained from natural products. This primary composition provides lycopene, particularly in a highly bioavailable and / or biologically effective state.

  In a preferred embodiment, the present invention provides a tomato extract or derivative thereof having an isomer ratio different from that of naturally occurring products so far available. In particular, the present invention relates to an extract or derivative having an E isomer content of 60% or less relative to the total lycopene content, preferably an E isomer content (according to HPLC) of 40% or less relative to the total lycopene content. About.

  In one embodiment, the present invention provides a primary composition comprising a particular combination of Z-isomers. Preferably, the Z / E isomer ratio in the primary composition of the invention should be greater than 1.

  Furthermore, the combination is preferably rich in 5-Z and 9-Z and poor in 13-Z. In a preferred embodiment, the amount of 5-Z and 9-Z is greater than 30%, preferably greater than 40%, most preferably greater than 50% relative to the total lycopene content. Also, the amount of 13-Z is less than 10%, preferably less than 5%, most preferably less than 3% based on the total lycopene content. Increasing certain 5-Z and 9-Z isomers and / or decreasing 13-Z isomers, for example, have higher bioavailability and higher biological effects A stable form of the primary composition can be obtained. Furthermore, the extract or derivative of the present invention is stable under normal storage conditions and is not reverse isomerized. Under normal protection conditions (no light and oxygen present), the total lycopene content and the E-isomer content remain constant. Even if the extract is kept at room temperature, the latter does not increase.

  Such a profile (ie low amount of 13-Z isomer of lycopene) can be achieved, for example, by isomerizing lycopene utilizing catalysis on a solid matrix such as clay or for extended periods of time. It can be obtained by heating.

  In one embodiment, the lycopene-containing material can be, for example, in the form of an extract, a concentrate or an oleoresin. In the context of the present invention, the term “oil-containing resin” means a lipid extract of lycopene-containing materials that may contain other carotenoids, triglycerides, phospholipids, tocopherols, tocotrienols, phytosterols and other less important compounds. Then it should be understood. Surprisingly, it has been found that the reverse isomerization of lycopene in isomerized tomato oleoresin can be minimized by reducing its content in the 13-Z isomer.

  In one embodiment, the lycopene-containing material can be an extract, concentrate or oleoresin obtained, extracted, enriched or purified from plant or vegetable material, microbial, yeast or animal origin products. . This is further subjected to treatment to increase the Z-isomer content of lycopene, as described below.

  If the source of lycopene is of plant origin, it may be vegetables, leaves, flowers, fruits and other parts of the plant. In a preferred embodiment, the source of lycopene is tomato (ie, whole tomato, tomato extract, tomato pulp, tomato puree, tomato skin, with or without seeds). Suitable plant or vegetable concentrates can be obtained, for example, by drying or lyophilizing freshly cut plants or vegetables, or their roots, fruits or seeds, and then optionally grinding or granulating the dried material. . Suitable methods for obtaining said plant or vegetable extracts are known in the art. The plant or vegetable extract is, for example, a freshly cut processed plant or vegetable, or its root, fruit or seed, water, one or more food solvents, or water and one or more foods. It can obtain by extracting using the mixture with the solvent for a use. Preferably, the extracts and concentrates of the present invention may be lipid or aqueous. Since carotenoids are fat-soluble, extraction with water removes unnecessary water-soluble components such as sugars, amino acids, soluble proteins and / or organic acids.

  When the lycopene-containing material is obtained from a microorganism, any microorganism that produces lycopene may be used, for example, a microorganism that works well for a specific body such as lactic acid bacteria. The product of animal origin may also be, for example, salmon, shrimp, krill or liver extract, or milk fraction. In this specification, the term “milk fraction” should be understood to mean any part of milk.

  In another embodiment, the lycopene-containing material can be an oleoresin. Suitable methods for obtaining oleoresin from the plant or vegetable are well known in the art. For example, the oil-containing resin can be obtained by lipid extraction using a solvent compatible with food, cosmetics or drugs. The oil-containing resin prepared by the conventional method has a lycopene content of about 0.05 to 50% by weight. The content of all E isomers of lycopene is usually greater than the Z isomer, for example, the Z / E isomer ratio of lycopene in the selected tomato oleoresin is about 7:93.

  The oleoresin is a preferred starting material for obtaining a primary composition according to the present invention because it contains antioxidants such as vitamin E or other carotenoids that also contribute to the stabilization of the composition. The biological activity and stability of the lycopene compound in the oleoresin can be particularly improved during the isomerization process, and the yield of Z-type lycopene in the primary composition can also be increased.

  The lycopene-containing material preferably comprises carotenes and xanthophylls such as zeaxanthin, astaxanthin, β-cryptoxanthin, capsanthin, canthaxanthin, lutein and derivatives thereof such as esters. The lycopene compound has been subjected to a treatment to increase the amount of Z-isomer in the primary composition.

  In order to obtain such isomeric profiles, lycopene-containing materials in the form of extracts, concentrates or oleoresin are neutral, acidic or basic solid catalysts (eg clays, zeolites, molecular sieves, ion exchangers) Is used to produce a mixture with a high Z / E ratio. The use of a solid catalyst to increase the Z-isomer lycopene is not contaminating and harmful to food, as the catalyst can be successfully removed by simple filtration or centrifugation. Also, geometric isomerization can be improved by a combination of the solid catalyst and other general means (for example, heat, light and radical initiator).

  In another embodiment, the extract or derivative according to the invention can be prepared starting from tomatoes, tomato parts (eg skin), derivatives (eg sauces and concentrates) or extracts. Isomerization is performed by heating for a long time in a solvent. This discovery is surprising considering the reverse technique derived from the prior art described in the “Background of the Invention” section.

  In particular, when tomatoes or derivatives thereof are used as starting materials, these can be treated with a solvent capable of extracting lycopene. The resulting extract is then heated to remove the solvent, thereby recovering the isomerized extract.

  On the other hand, if an extract or derivative is used as a starting material, it is taken up in a solvent and the mixture is heated for an appropriate time, then the solvent is removed, thereby recovering the isomerized extract. Solvents that can be used in the isomerization step are hydrocarbons, chlorinated hydrocarbons, esters, ketones, alcohols; specifically, C3-C10 aliphatic hydrocarbons, C1-C3 chlorinated solvents, C3-C6 esters, C3 -C8 ketones and C1-C8 alcohols; more specifically hexane, carbon tetrachloride, ethyl acetate, acetone and butanol. Isomerization in a solvent is carried out at a temperature in the range of 50 to 150 ° C, preferably in the range of 60 to 130 ° C. The isomerization time is in the range of 4 to 240 hours, preferably in the range of 10 to 180 hours.

  The Z / E isomer ratio in the primary composition is then increased to at least 20:80, preferably between 20:80 and 95: 5, more preferably between 30:70 and 90:10. Good. In a preferred embodiment, the (5Z + 9Z) / E ratio is greater than 1 and the 13Z form is partially removed.

  In one embodiment, the present invention provides a primary composition in the form of a powder, liquid or gel comprising a lycopene compound that has better bioavailability and / or biological effect than the compound alone. Also, the primary composition may be in a highly water dispersible composition state when choosing a powder state. In this case, the powder is dispersible in water at ambient temperature. The primary composition also provides a carotenoid that is particularly highly soluble in lipids and organic solvents, has a low tendency to crystallize, and has a low tendency to aggregate.

  In another embodiment of the invention, the primary composition may be used alone or in combination with other active compounds: vitamin C, vitamin E (tocopherol and tocotrienol), carotenoids (carotene, lutein, zeaxanthin) , Β-cryptoxanthin, etc.), ubiquinone (eg, CoQ10), catechin (eg, epigallocatechin gallate); coffee extract (eg, caffeol and caffeol) containing polyphenols and / or diterpenes, extract of chicory , Ginkgo biloba extract, proanthocyanidin rich grape or grape seed extract, spice extract (eg rosemary); soy extract containing isoflavones and related phytoestrogens and other flavonoid sources with antioxidant activity Fatty acids (e.g., n-3 fatty acids), phytosterols, prebiotic fibers, probiotic microorganisms, taurine, resveratrol, aminoacids, selenium, precursors of glutathione, or proteins, e.g., whey proteins.

  The primary composition can further comprise one or more emulsifiers, stabilizers and other additives. Emulsifiers that are compatible with the food sector are, for example, phospholipids, lecithin, polyoxyethylene sorbitan mono or tristearate, monolaurate, monopalmitate, mono or trioleate, mono or diglycerides. Any stabilizer known in the food, cosmetic or pharmaceutical field can be added. It is also possible to add flavorings, colorants and other suitable additives known in the food, cosmetic or pharmaceutical field. These emulsifiers, stabilizers and additives can be added according to the end use of the primary composition.

  In another aspect, the present invention provides an oral composition comprising a primary composition as described above in a food, food supplement, pet food product, cosmetic formulation or pharmaceutical formulation.

  In a preferred embodiment, the primary composition can be supplementarily added to a food composition for human consumption. The food composition may be, for example, a complete nutritional food, a dairy product, a chilled or shelfd beverage, a mineral water, a liquid beverage, a soup, a dietary supplement, an alternative food, a nutrition bar, a confectionery, milk or fermented milk Products, yogurt, milk powder, intestinal absorbable nutrition products, infant formula, infant nutrition products, cereal or fermented cereal products, ice cream, chocolate, coffee, culinary products such as mayonnaise, tomato puree Or salad dressing or pet food.

  For use in a food composition, the primary composition can be added to the food or beverage such that the daily intake of lycopene contained in the primary composition is about 0.001 to 50 mg. A daily intake of about 5-20 mg is considered preferred.

  Nutritional supplements for oral administration may be included in capsules, gelatin capsules, soft capsules, tablets, dragees, pills, pastes or troches, gums, or drinking solutions or emulsions, syrups or gels, about 0.001 of the primary composition. It is contained in an amount of ˜100% and can be taken directly with water or by other known means. The supplement may also contain sweeteners, stabilizers, additives, flavorings or colorants. Supplements for cosmetic purposes may further comprise compounds active on the skin. It should be understood that the supplement can be made by any method known to those skilled in the art.

  In another embodiment, the pharmaceutical composition comprising the primary composition is in an amount sufficient to at least partially capture the indication of the treatment or disease and its complications for prophylactic and / or therapeutic treatment Can be administered. In this specification, an amount sufficient to accomplish this is defined as "therapeutically effective amount". The effective amount for this depends on the severity of the disease and the weight and general condition of the patient.

  In prophylactic use, the primary composition of the invention can be administered to patients who are susceptible to or at risk of developing a particular disease. Such an amount is defined as a “prophylactically effective amount”. In this application, the exact amount will again depend on the patient's health and weight.

  In another embodiment, the primary composition of the invention can be administered with a pharmaceutically acceptable carrier, the nature of the carrier being of the mode of administration, eg parenteral, intravenous, oral and topical (including ocular). ) Depending on the route. The desired composition can be made using a variety of excipients including, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin cellulose, magnesium carbonate. The pharmaceutical composition may be a tablet, capsule, pill, solution, suspension, syrup, dry oral supplement or wet oral supplement.

  Preferably, for human use, the pharmaceutical composition of the present invention may comprise a predetermined amount of the primary composition as described above so that the daily lycopene amount is in the range of about 0.01-100 mg. When administered daily to pets, the amount of lycopene can range from about 0.01 to 100 mg.

  Those skilled in the art will consider, based on their knowledge, the intended tissue, e.g., skin, taking into account the route of administration, which may be injection, topical administration, intranasal administration, administration by implant or transdermal sustained release system, etc. It is understood that the appropriate ingredients and herbal form will be selected for targeting the active compound to the colon, stomach, kidney or liver.

In another embodiment, the present invention provides a cosmetic composition comprising the aforementioned primary composition. This is prepared, for example, in lotions, shampoos, creams, sunscreens, post-sun creams, anti-aging creams and / or ointments. Preferably, the content of the primary composition may be 10 ⁻¹⁰ to 10% by weight of the cosmetic composition. More preferably, the cosmetic composition comprises 10 ^{−8 to} 5% by weight of lycopene. Cosmetic compositions that can be used topically can further include fats or oils that can be used in cosmetics, such as those described in CTFA work, Cosmetic Ingredients Handbook, Washington.

  The cosmetic composition of the present invention may also contain other suitable cosmetic active ingredients. The composition further comprises a structuring agent and an emulsifier. Other excipients, colorants, flavorings or opacifiers can also be added to the cosmetic composition. This cosmetic product is understood to contain a mixture of different ingredients known to those skilled in the art, allowing the desired substance to penetrate rapidly into the skin and prevent deterioration during storage.

  It should also be understood that the concepts of the present invention apply as an adjunct therapy useful in currently used drug treatments as well. Since the primary compound of the present invention can be easily administered together with food materials, a special clinical diet containing a large amount of the primary composition is applied. After reading this specification in conjunction with the appended claims, it will be apparent to those skilled in the art that various different modifications can be made to the other aspects described herein.

  The present invention is further intended to protect skin tissue against aging, in particular to inhibit damage to the skin and / or mucous membrane by inhibiting collagenase and increasing collagen synthesis. It relates to the use of said primary composition, oral composition or cosmetic composition for the preparation of a product. In fact, the use of a primary composition as described above, for example, improves the bioavailability of lycopene compounds in the body and makes it possible to delay skin aging. The primary composition is also useful in the prevention or treatment of sensitive dry or reactive skin, or in improving skin density or hardness, improving skin photoprotection, cardiovascular disease or failure and cancer prevention or treatment . They have special advantages over hair and pet animal hair, such as improved hair or hair density, fiber diameter, color, oiliness, gloss and help prevent hair or hair loss.

  The positive effect of the primary composition of the invention on human or pet skin is, for example, minimal erythema dose (MED), colorimetric analysis, transepidermal water loss, DNA repair, measurement of interleukin and proteoglycan production, or collagen It can be measured using conventional methods such as enzyme activity, barrier function or cell regeneration, or ultrasonography.

(Example)
Example 1
Study of stability of lycopene isomers The stability of lycopene isomers was evaluated both in organic solvents and in tomato extracts.

material
Lycopene-rich tomato oleoresin was obtained from lndena spa (Milan, Italy). The total lycopene content was 9.1% and its total E and 5-Z isomers were 93.5% and 6.5%, respectively. Two types of isomerized oil-containing resins were prepared by heating a suspension of tomato oil-containing resin (1:10 w / w) in ethyl acetate for 1 hour or 48 hours. After cooling at room temperature, the suspension was centrifuged, and ethyl acetate in the collected supernatant was removed by distillation under reduced pressure.

  Di-t-butyl-hydroxy-toluene (BHT) and N-ethyldiisopropylamine were obtained from Fluka AG. All solvents were HPLC grade and were used without purification.

Isolation of pure lycopene isomers Pure 5-Z, 9-Z, 13-Z and all-E lycopene are collected by collecting the fractions containing the corresponding peaks after HPLC separation (see below for experimental conditions). Isolated from isomerized tomato oleoresin (heated for 1 hour). Peaks were collected during two consecutive HPLC runs and the corresponding fractions were stored.

Lycopene analysis Total lycopene amounts were determined using C ₁₈ precolumn (ODS Hypersil, 5 μm, 20 × 4 mm; Hewlett Packard, Geneva, Switzerland) and C ₁₈ column (Nova pak, 3.9 μm ID × 300 mm length, Millipore, Volketswil, Switzerland). In reverse phase HPLC. Separation at room temperature under isocratic conditions using a mobile phase consisting of acetonitrile / tetrahydrofuran / methanol / ammonium acetate 1% (533.5: 193.6: 53.7: 28, wt / wt / wt / wt) went. The mobile phase flow rate was 1.5 mL / min.

  Lycopene isomer profiles were obtained from Schierle et al. (Schierle, J., Bretzel, W., Buehler, I., Faccin, N., Hess, D., Steiner, K., Schuep, W. (1997). Food. Chem. 59: 459) as determined by normal phase HPLC. A sample of the isomerized oil-containing resin was dissolved in n-hexane containing 50 ppm BHT and rotated at maximum speed in an Eppendorf Lab centrifuge. The resulting supernatant was immediately analyzed by HPLC. The HPLC system used was a 1100 series Hewlett-Packard model and was equipped with a UV-visible diode array detector. Data were obtained simultaneously at 470 nm, 464 nm, 346 nm and 294 nm. Samples (10 μL) were separated using a combination of three Nucleosil 300-5 columns (4 mm ID × 250 mm length, Macherey-Nagel). Separation was performed at room temperature under isocratic conditions using a mobile phase consisting of n-hexane containing 0.15% N-ethyldiisopropylamine. The flow rate was 0.8 mL / min. Lycopene Z isomers were kinetics according to literature data.

  The amount of lycopene isomer was calculated based on the surface area of the HPLC peak using the same attenuation coefficient as all E-type lycopene. That is, since the attenuation coefficient of the Z isomer was found to be lower than that of all E isomers, the lycopene concentration in the product containing the Z isomer was estimated to be slightly lower.

Stability Test Conditions The stability of lycopene isomers was investigated both in tomato oleoresin and n-hexane isomerized by heating in ethyl acetate for 4 hours. For this purpose, pure lycopene isomers were stored in n-hexane for 33 days at room temperature in the absence of light, and isomerized tomato oleoresin was maintained at room temperature for 55 days in the absence of light. During storage, total lycopene concentration and lycopene isomer profile were measured at various time intervals.

(result)
Stability of lycopene isomers in n-hexane Table 1 shows the results of a stability test of pure lycopene isomers stored in n-hexane at room temperature in the absence of light. All isomers, including all E isomers, geometrically isomerized during storage. Form 13-Z is a poorly stable isomer: less than 50% of 5-Z, 9-Z and all E-type lycopene is converted after 33 days of storage, and more than 80% of 13-Z lycopene Converted to other isomers during the period. Also, the conversion pathway of 13-Z lycopene is different from other Z isomers: during storage in n-hexane, the 13-Z isomer is mainly converted to the all-E isomer, Z and 9-Z isomers were mainly converted to other Z isomers.

Stability of lycopene isomers in tomato oleoresin Table 2 shows the results of a stability test of lycopene isomers in tomato oleoresin heated in ethyl acetate for 48 hours.

  The total lycopene content was stable during storage at room temperature. However, the lycopene isomer profile changed significantly with decreasing 13-Z lycopene content and increasing total E lycopene. The content of 9-Z and 5-Z lycopene remained stable during storage.

(Conclusion)
Both stability studies showed that the 13-Z lycopene was much less stable than the 5-Z, 9-Z or all E isomers. As a result, isomerized tomato oleoresin with a low content of 13-Z lycopene should show good stability of its lycopene isomer profile.

(Example 2)
Isomerized tomato oleoresin with increased bioavailability Objective The purpose of this experiment was to investigate the bioavailability of various Z lycopene isomers in humans. In order to clarify the bioavailability of specific Z lycopene isomers in humans, the different Z lycopene isomer content of tomato oleoresin was increased to about 60% of the total lycopene content. That is, one is rich in 5-Z lycopene, the other is rich in 13-Z lycopene, and the last is rich in a mixture of 9-Z and 13-Z lycopene.

(Materials and methods)
Subjects 30 healthy men were enrolled in the study. Inclusion criteria were that the subject was not vegetarian and not a smoker and had no metabolic disease such as diabetes; hypertension; kidney, liver or pancreatic disease; or ulcer. The subject was not afflicted with lipid disease, that is, the ratio of plasma cholesterol to HDL cholesterol was less than 5.0 and the crystalline triacylglycerol (TAG) concentration was less than 1.5 mmol / L. Due to the large amount of blood drawn during the study, subjects needed to have blood hemoglobin concentrations above 13 g / dL. If you use drugs that change cholesterol levels, treatments that lower blood lipids, or vitamin and mineral supplements, or have undergone major gastrointestinal surgery 3 months before the start of the study and until the completion of the study; Subjects were excluded from the study if they exercised vigorously; and consumed more than 2 glasses (3 dL) of wine, more than 2 glasses of beer (3 dL) or more than 1 glass of strong liquor (shot glass) per day. Twenty-seven of the 30 volunteers completed four types of post-meal testing. Three volunteers abandoned the study prior to termination for the following reasons: inability to participate, medical treatment of eye wounds, nausea due to consumption of fat diet. The subject was 24 ± 1 years old, had a body weight of 70 ± 1 kg, and had a body mass index (BMI) of 22.5 ± 0.3 kg / cm ² .

  The protocol was approved by the Ethics Committee of Marseille (Marseille, France). Subjects received information about the research background and design and submitted written informed consent prior to participation. They were free to withdraw from research at any time.

Study Design This was double-blind, randomized, comprised of 4 phases, tried 4 overlapping clinical treatments and provided a washout period of at least 3 weeks. After fasting overnight, subjects arrived at the Clinical Pharmacology and Therapeutic Trial Center of the University of Marseille, a standard diet consisting of 25 mg lycopene mixed with 40 g peanut oil mixed with 70 g wheat semolina (cooked with 200 mL tap water). Consumed. In addition, they ate 125 g of yogurt containing 40 g of bread, 60 g of cooked egg white and 5 g of white sugar and drank 330 mL of water (Aquarel, Nestle). This standard diet provided 842 kcal (3520 kJ) with the following nutritional composition. Protein (11.7%), carbohydrate (39.3%) and lipid (49.0%). This meal was consumed within 15 minutes. No other meals were given over the next 6 hours, but a bottle (330 ml) of water was allowed for 3 hours after absorption (Aquarel, Nestle).

Lycopene supplements Four different tomato products were tested for each total lycopene amount of 25 mg. These consist of:

・ Tomato paste containing lycopene, mostly all-E (Thorny, Switzerland)
Tomato oleoresin rich in 5-Z lycopene Tomato oleoresin rich in 13-Z lycopene Tomato oleoresin rich in a mixture of 9-Z and 13-Z lycopene Table 3 shows the production of these four types of tomatoes The lycopene content of the product and the lycopene isomer profile are shown.

Collection of blood samples Fasting blood was drawn from the elbow vein by venipuncture into a vacuum tube containing potassium EDTA / K3, which was immediately placed in an ice-water bath and covered with aluminum foil to avoid exposure. Fasting blood samples were taken before consumption of the standard diet, ie, 20 and 5 minutes before and 2 hours, 3 hours, 4 hours, 5 hours and 6 hours after absorption. Tubes containing blood were protected from light, stored at 4 ° C., and then centrifuged within 2 hours (10 minutes, 4 ° C., 2800 rpm) to separate plasma. Inhibitor (10 μL / mL) was added (Cardin et al., Degradation of apolipoprotein B-100 of human plasma low density lipoproteins by tissue and plasma kallikreins, Biol Chem 1984; 259: 8522-8.).

Isolation of plasma triglyceride-rich lipoprotein (TRL) After consumption of a fat diet, the lipophilic molecules of the food are taken up by chylomicrons secreted into the blood. Lipoproteins are separated by ultracentrifugation based on density. Due to the fairly similar density of chylomicron (0.95 g / ml) and VLDL (1.006 g / ml), it is impossible to separate one from the other and it is collected together in one fraction. Called triglyceride-rich lipoprotein (TRL). However, in the postprandial state, this plasma TRL fraction mainly comprises chylomicrons secreted from the intestine, thus providing an excellent assessment of intestinal bioavailability.

  Triglyceride-rich lipoprotein (TRL) containing mostly VLDL, mainly containing chylomicron, was immediately isolated by ultracentrifugation as follows: 6 mL of plasma was placed with 0.9% NaCl solution and more than L7 Ultracentrifugation was performed in a SW41TI rotor (Beckman) for 28 minutes at 10 ° C. and 32000 rpm in a centrifuge (Beckman). Immediately after centrifugation, TRL was collected and stored at −80 ° C. and analyzed. Lycopene analysis was performed within 10 days and triacylglycerol was analyzed within 30 days.

Analytical determination Triglycerides were evaluated by enzymatic and colorimetric methods using a commercial kit (Kit Bio-Merieux).

  Total lycopene and lycopene isomer profiles were determined by reverse-phase HPLC and normal-phase HPLC methods (M. Richelle, K. Bortlik, S. Liardet, C. Hager, P. Lambelet, LA Applegate, EA Offord, respectively). J. Nutr. (2002) 132, 404-408.). Total lycopene content was calculated as the sum of 5-Z, 9-Z, 13-Z, xZ and all E-type lycopene isomers. Lycopene isomers were quantified using the attenuation coefficient of all E-type lycopene. This is because the exact values of all individual Z-type lycopenes are still unknown. The profile of lycopene isomers is determined by the ratio expressed as a percentage of the individual lycopene isomers relative to the total lycopene.

Statistical analysis Lycopene bioavailability was assessed by measuring the area under the lycopene concentration-time curve in TRL (AUC). This area was calculated over a period of 0-6 hours using the trapezoidal method (AUC (0-6 hours)). Data are presented as mean ± SEM. Baseline concentration is the average of the concentrations measured in two plasma samples taken before consuming a standard diet containing 25 mg of lycopene from tomato matrix. For each subject and each lycopene treatment, AUC (0-6 hours) was calculated by subtracting the baseline concentration from the concentration value measured at each time point after absorption. If this value was negative, it was considered zero.

  For each treatment, if the distribution of (AUC (0-6 hours)) is normal (Skewness and Kurtosis tests) with or without logarithmic transformation, the treatment as a fixed effect and the subject as a random effect Comparisons were made using a linear mixed model using.

  All statistical analyzes were performed using SAS software (version 8.2; SAS Institute, Cary, NC). The rejection level in the statistical test was equal to 5%.

(result)
Lycopene bioavailability As the four tomato treatments induced changes in the degree of triglyceride secretion, triglyceride absorption was used to normalize lycopene bioavailability (AUC (0-6 hours)) .

  Normalized lycopene bioavailability was significantly different between the four tomato treatments (FIG. 1).

  Surprisingly, lycopene bioavailability from 5-Z lycopene-rich tomato oleoresin has three other treatments: tomato paste, 13-Z lycopene-rich tomato oleoresin, and About 2 times better than that from tomato oleoresin rich in a mixture of 13-Z and 9-Z lycopene (p <0.0001) (FIG. 1).

  However, lycopene showed bioavailability from tomato paste to the same extent as from a mixture of 13-Z and 9-Z type tomato oleoresin.

  Lycopene appearing in the 13-Z type tomato oleoresin showed a slight but significantly lower bioavailability compared to tomato paste (p <0.03).

(Conclusion)
These results indicate that the structure of the lycopene molecule significantly affects lycopene traffic in the gastrointestinal tract, and consequently the amount of lycopene absorbed. Lycopene bioavailability from tomato extract rich in 5-Z lycopene is about twice that from tomato paste. In contrast, lycopene present in tomato extracts rich in a mixture of 9-Z and 13-Z lycopene is as bioavailable as that present in tomato paste, whereas 13- Tomato oleoresin rich in Z-type lycopene shows lycopene with a little less bioavailability. Several authors have already pointed out that the presence of Z-type lycopene in tomato products is associated with increased lycopene bioavailability. The present invention shows that the improvement in lycopene bioavailability is specifically related to the structure of lycopene, ie 5-Z lycopene> 9-Z lycopene> 13-Z lycopene. This is the first study to show.

(Example 3)
Extraction and isomerization in ethyl acetate 52 kg of fresh tomatoes containing 100 ppm of lycopene are cut and homogenized. A part of the water is evaporated off under reduced pressure to obtain 18 kg of tomato concentrate. This is extracted with 36 L of water-saturated ethyl acetate, the extraction keeping the mixture at room temperature, shielding from light and stirring for 2 hours. The extract is then separated from the tomato concentrate. The procedure is repeated twice with a total of 108 L of solvent on such tomato concentrate. The combined extracts are washed with 27 L of water in a separatory funnel. The aqueous phase is then discarded and the organic phase is concentrated under reduced pressure to give a suspension containing 10% w / v dry residue. The dry residue has a total lycopene content of 9.1% w / w and a Z-isomer content of 0.46% w / w. The mixture is refluxed (76 ° C.) for 7 days with stirring and then concentrated to dryness under reduced pressure.

  46.8 g of a final extract having a total lycopene content of 9% w / w and a Z-isomer content of 5.59% w / w is obtained, in particular, an E-isomer content of 3.41% w. / W and the 13-Z isomer content is 0.16% w / w. The HPLC profile of the extract is reported in FIG.

Example 4
Extraction and isomerization in hexane 10 kg of fresh tomatoes containing 140 ppm of lycopene are cut and homogenized. A portion of the water is removed by evaporation under reduced pressure to give 2.5 kg of tomato concentrate, which is extracted with 12.5 L of hexane. Extraction is carried out for 2 hours with stirring, keeping the mixture at room temperature, protected from light. The extract is then separated from the tomato concentrate. The procedure is repeated once for such tomato concentrate using a total of 25 L of solvent. The extracts are combined and concentrated under reduced pressure to give a solution containing 10% w / v dry residue. The dry residue has a total lycopene content of 9.1% w / w and a Z-isomer content of 0.46% w / w. The mixture is refluxed (69 ° C.) for 6 days with stirring and then concentrated to dryness under reduced pressure. 16.5 g of a final extract having a total lycopene content of 9.1% w / w and a Z-type isomer content of 5.62% w / w is obtained, in particular an E-type isomer content of 3.38. % -W / w and the 13-Z isomer content is 0.18% w / w.

(Example 5)
Isomerization in butanol Cut and homogenize 10 kg of fresh tomatoes containing 90 ppm lycopene. A portion of the water is removed by evaporation under reduced pressure to give 3.4 kg of tomato concentrate, which is extracted with 7 L of water saturated ethyl acetate. Extraction is carried out for 2 hours with stirring, keeping the mixture at room temperature, protected from light. The extract is then separated from the tomato concentrate. The procedure is repeated twice for such tomato concentrate using a total of 21 L of solvent. The combined extracts are washed with 5.3 L of water in a separatory funnel. The aqueous phase is then discarded and the organic phase is concentrated to dryness under reduced pressure. A dry residue (9.8 g) with a total lycopene content of 7.8% w / w and a Z-isomer content of 0.40% w / w is suspended in 98 ml of n-butanol. The mixture is maintained at 130 ° C. with stirring for 4 hours and then concentrated to dryness under reduced pressure. 9.8 g of a final extract having a total lycopene content of 6.35% w / w and a Z-isomer content of 4.50% w / w is obtained, in particular, an E-isomer content of 1.85 % -W / w and the 13-Z isomer content is 0.47% w / w.

(Example 6)
Isomerization over solid catalyst Material Lycopene-rich tomato oleoresin was obtained from lndena spa (Milan, Italy). Its total lycopene content was 9.1%, of which the total E and 5-Z isomers were 93.5% and 6.5%, respectively.

Method A suspension of tomato oleoresin in ethyl acetate (1: 100 w / w) was filtered and incubated with 5% solid catalyst at room temperature with constant agitation for 2 hours. The mixture was centrifuged at maximum speed in an Eppendorf Lab centrifuge and a portion of the supernatant was evaporated under N ₂ and resuspended in n-hexane / BHT.

Lycopene Analysis Total lycopene content and lycopene isomer profile were determined by reverse phase HPLC and normal phase HPLC, respectively, under the analytical conditions described in Example 1.

(result)
The lycopene isomer profile measured in tomato oleoresin isomerized for 2 hours at room temperature with solid catalyst is reported in Table 4.

  Lycopene was efficiently isomerized during 2 hours of reaction in ethyl acetate at room temperature in the presence of Tonsil Optimum or Amberlyst 15. When both catalysts were used, a large amount of lycopene all E isomer was converted to Z isomer. Of the identified lycopene isomers, the 5-Z form was predominantly formed, followed by the 9-Z and 13-Z forms, respectively. That is, the concentration of the 13-Z type isomer was less than 10% in the isomerized tomato oleoresin.

Claims (6)

  A method for producing a stable composition rich in cis-lycopene (Z-isomer) by heating a lycopene-containing material in a solvent for a long time.   The method according to claim 1, wherein the lycopene-containing material comprises tomato, a part of tomato, a derivative thereof or a tomato extract in a solvent.   The process according to claim 1, wherein the solvent is a C3-C10 aliphatic hydrocarbon, a C1-C3 chlorinated solvent, a C3-C6 ester, a C3-C8 ketone or a C1-C8 alcohol.   4. A process according to claim 3, wherein the solvent is selected from hexane, carbon tetrachloride, ethyl acetate, acetone and butanol.   The method according to claim 1, wherein the heating temperature is in the range of 50 to 150 ° C.   The method according to claim 1, wherein the heating time is in the range of 4 to 240 hours.

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